Hemin, which gets released during erythrocyte lysis, induces concentration-dependent platelet activation, aggregation, and thrombus formation as well as plasma membrane destruction and cytoskeleton reorganization. Classical platelet antagonists do not inhibit the plasma membrane destruction induced by high concentrations of hemin. Both cyclic guanosine monophosphate (cGMP) and adenosine 3':5' -cyclic monophosphate (cAMP) are key endogenous inhibitors of platelet activation. Thus, we investigated whether they inhibit hemin-induced plasma membrane destruction, both individually and in combination.In addition to standard platelet assays, we performed cGMP and cAMP ELISAs, immunoblot analysis of vasodilator-stimulated phosphoprotein (VASP) and immunofluorescence staining. We found that pharmacological modulation of these pathways via NO donors (DEA/NO), soluble guanylyl cyclase (sGC) stimulators (riociguat) or IP receptor agonists (PGE1), and phosphodiesterase (PDE) inhibitors (PDE-5 inhibitor: sildenafil, PDE-3 inhibitor: ibudilast), phosphorylate the downstream vasodilator-stimulated phosphoprotein (VASP) and inhibit hemin-induced platelet activation and degranulation. In particular, synergistically modulation of cGMP and cAMP and the resulted phosphorylation of VASP at Ser239 and Ser157 significantly attenuates platelet aggregation and plasma membrane destruction induced by high concentrations of hemin. Further, the riociguat NO-induced cGMP synthesis was significantly reduced in the presence of hemin. In comparison, the PGE1-induced cAMP synthesis was enhanced in the presence of hemin. In conclusion, high concentrations of hemin change the cGMP and cAMP synthesis induced by their associated stimulators, while promoting plasma membrane destruction, which can be significantly inhibited by the simultaneous administration of riociguat DEA/NO and PGE1.

Fracture healing failure remains a major complication in trauma and orthopedic surgery. The transplantation of autologous cancellous bone grafts represents the gold standard for the treatment of atrophic non-unions. However, during revision surgery the grafts can be exposed to a significant period of intraoperative ischemia, which may have detrimental effects on their quality and functionality. Therefore, we analyzed the effects of different periods of ischemia (0, 30, 60 and 90 min) on cellular stress, gene expression and viability of the bone grafts, to determine a critical ischemia time window for transplantation. Graft samples were harvested from 24 patients undergoing revision surgery due to bone healing failure. Analyses included mRNA profiler arrays, reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. Ischemia lasting 60 min or longer induced the expression of stress-induced genes, such as JUN and DUSP1. This was associated with early cellular stress within the grafts, as indicated by the presence of hypoxia-inducible factor (HIF)-1α-positive cells and an increased number of senescent p16-positive cells at early time points of ischemia. Additional analyses revealed a significantly higher number of apoptotic cleaved caspase-3-positive cells at 60 and 90 min of ischemia, demonstrating a compromised viability of the grafts. Moreover, RT-PCR analyses revealed a shift from a pro-osteogenic towards a pro-chondrogenic extracellular matrix (ECM) gene expression profile. Taken together, periods of ischemia of 60 min or longer after tissue harvesting should be avoided during cancellous bone graft transplantation to preserve graft viability.

Achieving complete cardiac ablation requires higher electric fields, increasing the risk of vasospasm or collateral injury. A potential strategy to enhance ablation efficacy without raising field strength is selective pharmacological targeting of reversibly electroporated cells. Gelonin, a type I ribosome-inactivating protein, inhibits protein synthesis once inside the cytosol but cannot cross intact plasma membranes. We investigated whether transient membrane permeabilization during PEF exposure allows intracellular gelonin delivery to enhance cardiomyocyte ablation. Gelonin cytotoxicity was assessed via long-term live-cell imaging. Cardiomyocyte monolayers were exposed to μsPEF (20 × 100 μs pulses at 1 Hz) or nsPEF (200 × 300 ns pulses at 10 Hz) in 0-150 nM gelonin using contact electrodes positioned by an automated system. Cell death was quantified by propidium iodide uptake, caspase-3/7 activity monitored apoptosis. Gelonin alone was non-toxic. Simulations showed adsorption without membrane penetration. Under μsPEF, gelonin reduced LD₅₀ from 0.9 to 0.6 kV/cm (p < 0.0001), corresponding to a 33% reduction in the electric field strength required to induce 50% cell death. In contrast, LD₅₀ of nsPEF protocol was not significantly affected by gelonin (p = 0.13). Gelonin selectively enhances μsPEF-mediated cardiomyocyte death, representing a strategy to improve ablation efficacy while limiting reversible electroporation.

Abstract Background and Aims Acute myocarditis (AM) is a T cell-mediated myocardial disease with clinical manifestations ranging from mild chest pain to cardiogenic shock. Reliable biomarkers to stratify patients and guide therapy are currently lacking. In particular, the extent of the dysregulation of inflammatory pathways, and the impact on myocardial dysfunction, remain elusive. Methods Serum analyses were performed in prospectively recruited AM patients (n = 103) from two independent cohorts. Multimodal data integration combining profiling of cytokine and chemokine dysregulation with clinical biomarkers was used to define clinical phenotypes with distinct inflammatory signatures. Machine-learning and regression models were applied to determine biomarkers that indicate clinical severity. Results Immuno-proteomic profiling revealed conserved inflammatory patterns across AM cohorts, dominated by T cell-related cytokines and chemokines. In addition, AM patients showed dysregulation of fibroblast-derived cytokines, including hepatocyte growth factor (HGF), bone morphogenic protein 4 (BMP4) and the BMP4 inhibitors Gremlin-1 (GREM1) and Gremlin-2 (GREM2). Data integration and unsupervised clustering revealed two immuno-clinical phenotypes, linking T cell activation and fibroblast dysregulation to disease severity. Machine learning-based analysis identified CXCL10, GREM2 and LVEF as critical parameters for stratifying disease severity. Conclusions These findings highlight a systemic T cell activation signature as diagnostic hallmark of AM. In addition, dysregulation of fibroblast-derived tissue cytokines serves as an indicator for distinct immuno-clinical phenotypes in myocardial inflammatory disease. Thus, the clinically relevant link between T cell-driven immune activation, myocardial inflammation and fibroblast-driven remodelling provides a versatile set of parameters to identify severe manifestations of AM. Graphical Abstract Key Question: Are serological immune signatures linked to clinical severity in acute myocarditis and do they enable patient stratification? Key Findings: T cell- and fibroblast associated proteomic signatures indicate disease severity in acute myocarditis. Novel immuno-clinical phenotypes stratify patients according to distinct immune responses and clinical manifestations. CXCL10, GREM2 and LVEF are the most important parameters to identify immuno-clinical phenotypes. Take Home Message: CXCL10, GREM2 and LVEF emerge as key determinants for a severe immuno-clinical phenotype in acute myocarditis, highlighting the role of T cell-fibroblast interaction in the disease process and linking T cell activation, fibroblastic tissue remodelling and impaired cardiac function.

Resident cardiac fibroblast–derived (RCF-derived) cardiac myofibroblasts (CMFs) contribute to myocardial repair but also drive adverse ventricular remodeling and contractile dysfunction after myocardial infarction (MI). The sodium-activated potassium channel Slick (Slo2.1) has been described in cardiomyocyte (CM) mitochondria; however, transcriptomic analyses indicate higher Slick expression in RCFs/CMFs. Here, we investigated the role of Slick in cardiac fibroblast function and post-MI remodeling. Using live-cell imaging and whole-cell patch-clamp recordings, we found that plasma membrane Slick channels in RCFs and CMFs regulated potassium (K + ) efflux and modulated store-operated calcium entry (SOCE), particularly in CMFs. Global Slick KO and conditional CMF-specific KO hearts exhibited reduced fibrosis and preserved left ventricular function after ischemia/reperfusion injury. This cardioprotection was associated with diminished CMF activation and proliferation, reduced inflammation, and improved CM survival after MI. Collectively, these findings identify fibroblast Slick channels as regulators of SOCE-dependent fibrogenesis and demonstrate that their deletion mitigates maladaptive remodeling and functional decline after MI.

BACKGROUND:Risk stratification in non-ischemic cardiomyopathies (NICM) remains challenging despite guideline-based phenotypic classification using multimodal diagnostics including endomyocardial biopsy (EMB). We aimed to identify EMB-derived histological and molecular markers that improve phenotypic characterization and long-term risk stratification in patients with NICM. METHODS:In this prospective cohort study, 703 consecutive patients with symptomatic NICM underwent standardized multimodal evaluation, including clinical assessment, cardiac imaging, and endomyocardial biopsy. Biopsy specimens were analyzed using histology, immunohistochemistry, and targeted myocardial mRNA profiling. Associations between endomyocardial markers, and fibroinflammatory remodeling, imaging parameters, and molecular signatures were assessed cross-sectionally. Long-term prognostic relevance was evaluated using survival and multivariable prediction analyses during follow-up of up to fifteen years for all-cause mortality, cardiovascular mortality, implantable cardioverter-defibrillator (ICD) implantation, and appropriate ICD discharge. RESULTS:Elevated myocardial Gremlin-1 expression was associated with increased fibrosis, adverse cardiac remodelling, reduced left ventricular function, and enrichment of pro-fibrotic and inflammatory mRNA signalling pathways. Myocardial and circulating Gremlin-1 expression was independently associated with all-cause and cardiovascular mortality, and ICD implantation and discharge. Machine learning-based phenotyping using histological EMB data identified Gremlin-1 as a key predictive feature of poor prognosis. Incorporation of Gremlin-1 into predictive models significantly improved long-term cardiovascular risk stratification in NICM patients. CONCLUSION:Our results unveil that Gremlin-1 is associated with inflammation and cardiac remodelling in patients with NICM, and patients with Gremlin-1+ EMB and high plasmatic Gremlin-1 concentrations are at elevated risk to develop adverse cardiovascular events. Thus, the histological evaluation of Gremlin-1 may help to improve risk discrimination and management of NICM and HF patients.

BACKGROUND:Inflammation orchestrates an outcome after acute myocardial infarction (AMI). Thromboinflammation, via the CD40- and CD40 ligand (CD40L)-mediated platelet-leukocyte interaction, is involved in post-AMI inflammation. OBJECTIVES:This study hypothesized that acetylsalicylic acid (ASA) exerts pleiotropic cardioprotective effects beyond prevention of reinfarction by reducing thromboinflammation and infarct size. METHODS:A murine AMI model was used to investigate the effects of low-dose ASA, which is applied preischemia or after induction of ischemia (intraischemia), on post-AMI thromboinflammation and the outcome. To investigate the underlying mechanisms, platelet and neutrophil depletion and genetically induced and antibody-induced CD40L deficiency were applied. Thromboinflammation markers were analyzed. Translationally, the outcome after ST-elevation myocardial infarction (STEMI) was measured in ASA-pretreated vs ASA-naive patients (ClinicalTrials.gov ID: NCT03539133). RESULTS:Both ASA treatment preischemia and intraischemia reduced infarct size and thromboinflammation and improved cardiac function and remodeling. The scar size was smaller with ASA preischemia 21 days after AMI but not with ASA intraischemia. This cardioprotection was blunted in the absence of (a) platelets or (b) neutrophils. Both pharmacologic inhibition or genetic deficiency of CD40L abrogated ASA's protective effect. Accordingly, ASA-pretreated patients with STEMI had improved outcome (12.5% vs 23.8%; hazard ratio, 0.50; 95% CI, 0.31-0.80; P < .001). This was driven by reduced mortality without differences in recurrent AMI. CONCLUSION:Existing ASA therapy shows pleiotropic effects in the reduction of thromboinflammation and improvement of outcome after AMI, independent of its effects on the occurrence of ischemia itself. This should be considered while choosing timing of initiation and the optimal antithrombotic regime post-AMI in patients with coronary artery disease.

AIMS:Platelets play a major role in thrombo-inflammatory cardiovascular diseases such as myocardial infarction. Although platelet function is crucially determined by kinases, the impact of Casein Kinase 2α (CK2α) on platelet activation during arterial thrombosis and myocardial remodeling following ischemia and reperfusion (I/R) injury is not known. METHODS AND RESULTS:Using platelet-specific deletion of Csnk2a1 in mice, the evaluation of the CK2α-dependent platelet phosphoproteome revealed a diminished phosphorylation of the IP3 receptor type-1 in Csnk2a1-deficient mice. This finding was accompanied by attenuated IP3-induced Ca2+ mobilization, impaired integrin αIIbβ3 activation, abrogated platelet aggregation and secretion, as well as defective spreading on fibrinogen in response to collagen-related peptide. Accordingly, without affecting primary hemostasis, thrombotic vascular occlusion in vivo was diminished in Csnk2a1-deficient mice. When subjected to a myocardial I/R injury model, these mice displayed improved cardiac outcome when compared with wildtype mice. Raman spectromics, spatial metabolomics and molecular approaches revealed locally a CK2α-dependent release of chondroitin sulfate and transforming growth factor-β from platelets, which was associated with significantly reduced ventricular fibrosis and improved heart function in Csnk2a1-deficient mice. CONCLUSION:Altogether, our results disclose CK2α as pivotal player in platelet activation and pathogenesis of post-ischemic myocardial remodeling, including myocardial fibrosis and left ventricular impairment following myocardial ischemia.

Neutrophil extracellular trap formation (NETosis) affects a wide variety of clinically relevant human diseases. Although lipid remodeling is essential for neutrophil function and membrane rupture during NETosis, the neutrophil lipidome and its dynamics have not been characterized. Thus, we establish a quantitative lipidome of human neutrophils comprising 1048 species across nine orders of magnitude and map its remodeling during NETosis. NET formation caused profound alterations in the phosphatidylinositol, phosphatidic acid, diacylglycerol (DG), and lyso-glycerophospholipid levels. Calcium- and reactive oxygen species-dependent NETosis pathways displayed distinct lipidomic trajectories yet converged on the significance of phospholipid lipase networks. Pharmacological inhibition of this networks altered lipid composition and markedly impaired NETosis, while DG treatment revoked the effect. Together, our findings reveal lipid remodeling as a fundamental determinant of NETosis and identify interconnected and dependent phospholipid lipase networks with downstream DG-dependent signaling as a potential therapeutic target in NET-associated diseases.

BACKGROUND Nanosecond pulsed electric fields (nsPEFs) are a promising method for cardiac pulsed field ablation, currently in early clinical trials. However, effective ablation often requires high voltages, more pulses, and higher frequencies, which can raise tissue temperatures because of Joule heating. Fractionated pulse delivery can help mitigate thermal effects and potentially evoke electrosensitization, increasing cell damage. OBJECTIVE This study evaluates the effects of fractionated nsPEF on treatment efficacy and its selectivity against cardiomyocytes, aiming to determine whether fractionation improves ablation outcomes. METHODS Monolayers of HL-1 murine cardiomyocytes, MHEC5-T murine endothelial cells, AC16 human cardiomyocytes, and human umbilical vein endothelial cells were exposed to pulsed electric fields using a contact electrode operated by a custom robotic system. Cell viability and permeability were measured using wide-field fluorescence microscopy. Stained areas were matched to simulated electric fields for dose-response curves. Fractionation effects were also validated in an ex vivo murine model. RESULTS Fractionation of nsPEF reduced the electric field affecting 50% of cells for plasma membrane permeabilization by 10% compared with a single train of 200 pulses (P < .0001). This translated into enhanced cardiomyocyte ablation, with fractionated exposure lowering the electric field affecting 50% of cells for cell killing by 13% (P < .0001). Ex vivo results further confirmed a larger ablation area with fractionated nsPEF (P < .0001). CONCLUSION Fractionated nsPEF improves cardiac ablation efficiency by enhancing membrane permeability and cell-killing effect. These findings suggest that fractionated delivery could optimize nsPEF therapies, offering a more effective approach for cardiac ablation.

ABSTRACT:Throughout thrombopoiesis megakaryocytes (MKs) form proplatelets within the bone marrow (BM) and release platelets into BM sinusoids. Casein kinase 1α (CK1α) is a major player and thus, an important therapeutic target in several hematological malignancies. This study aimed to define the role of CK1α for the essential steps of thrombopoiesis and to dissect potential mechanisms of thrombocytopenia. MK-specific CK1α-deficiency resulted in a macrothrombocytopenia. Ck1αPf4Δ/Pf4Δ mice displayed a substantial BM hyperplasia with pivotal changes in MK nuclear lobulation and reduced contact to BM sinusoids. Ck1αPf4Δ/Pf4Δ MKs displayed a defective cytoskeleton organization reflected by a decreased amount of polymerized filamentous actin and disturbed microtubule dynamics due to p21/p53 accumulation and impaired Rho-associated protein kinase (ROCK)/LIM domain kinase (LIMK)/cofilin signaling. Further, pronounced defects in DMS (demarcation membrane system) polarization and proplatelet formation of Ck1αPf4Δ/Pf4Δ MKs, unraveled CK1α as a prerequisite for thrombopoiesis. Our findings could be translated into a human approach, because a CRISPR/Cas9-mediated genetic deletion of CSNK1A1 in MKs derived from human CD34+ progenitor cells resulted in a substantial defect in human MK maturation and platelet production. The present observations elucidated CK1α as an important signaling molecule in MK cytoskeletal dynamics and polarization, proplatelet formation, and polyploidization, thus highlighting the crucial role of CK1α in platelet biogenesis.

AIMS:Pulsed field ablation (PFA), a cardiac ablation technique using microsecond pulsed electric fields (µsPEF), is widely used in clinical settings, while nanosecond pulsed electric fields (nsPEF) have recently entered clinical trials. Selective ablation of cardiomyocytes over endothelial cells is critical to prevent adverse remodelling, arrhythmias, and thrombosis, yet comparative data on nsPEF vs. µsPEF remain limited. This study investigates the cytotoxic effects and cell death mechanisms induced by nsPEF and µsPEF in cardiac and endothelial cells. METHODS AND RESULTS:Human cardiomyocytes and endothelial cells were exposed to varying electric field intensities with nsPEF and µsPEF using custom-built automated setup to assess permeabilization and cell death. Raman spectroscopy evaluated biochemical changes in cardiomyocytes following electroporation. Ex vivo epicardial ablation was performed on murine hearts using customized electrodes. Maximal cardiomyocyte death occurred 24 h after both pulse types in vitro. Ex vivo, both pulse types produced visible myocardial lesions as early as 1 h post-exposure, with lesion size progressively increasing up to 4 h. Microsecond pulsed electric fields induced significantly greater endothelial damage (ED50: 1.18 kV/cm) than damage to cardiomyocytes (ED50: 1.28 kV/cm), whereas nsPEF affected both cell types equally (ED50: 7.27 kV/cm vs. 7.24 kV/cm). Raman spectroscopy analysis of exposed cells indicated that µsPEF predominantly triggered necrotic or unregulated cell death, while nsPEF exposure was associated with regulated, apoptotic cell death. CONCLUSION:Pulse duration critically determines electroporation selectivity and downstream death pathways. Nanosecond pulsed electric fields favoured regulated cell death and cardiomyocyte selectivity, highlighting its potential to improve the safety and durability of PFA.

High-risk acute pulmonary embolism (PE) is a life-threatening condition necessitating hemodynamic stabilization and rapid restoration of pulmonary perfusion. In this context, evidence regarding the benefit of advanced circulatory support and pulmonary recanalization strategies is still limited. In this observational study, we assessed data of 1060 patients treated for high-risk acute PE with 991 being included in a target trial emulation to investigate all-cause in-hospital mortality estimates with different advanced treatment strategies. The four treatment groups consisted of patients undergoing (I) veno-arterial extracorporeal membrane oxygenation (VA-ECMO) alone (n = 126), (II) intrahospital systemic thrombolysis (SYS) (n = 643), (III) surgical thrombectomy (ST) (n = 49), and (IV) percutaneous catheter-directed treatment (PCDT) (n = 173). VA-ECMO was allowed as bridging to pulmonary recanalization in groups II, III, and IV. Marginal causal contrasts were estimated using the g-formula with logistic regression models as the primary approach. Sensitivity analyses included targeted maximum likelihood estimation (TMLE) with machine learning, inverse probability of treatment weighting (IPTW), as well as variations of estimands, handling of missing values, and a complete target trial emulation excluding the VA-ECMO alone group. In the overall target trial population, the median age was 62.0 years, and 53.3

Autosomal dominant mutations in ELANE (elastase, neutrophil expressed) cause severe congenital neutropenia (CN) and cyclic neutropenia (CyN). Inhibiting ELANE expression, either by CRISPR-Cas9-mediated ELANE knockout or promoter targeting using CRISPR-Cas9 nickase, has emerged as a promising gene therapy strategy to restore defective granulocytic differentiation of transplantable hematopoietic stem cells from CN patients. We developed an adenine base editor (ABE)-mediated approach targeting two nucleotides in the ELANE promoter to suppress neutrophil elastase expression, called PRECISE. Analysis of mRNA- and protein-based delivery of ABE revealed that although both platforms were effective in editing hematopoietic stem and progenitor cells from healthy donors with over 80% editing, only protein-based ABE delivery achieved over 68% editing in CN patient cells. Interestingly, 10%-19% editing in CN patients' hematopoietic cells using ABE mRNA restored their granulocytic differentiation in vitro, with a marked expansion and differentiation of ABE ribonucleoprotein (RNP)-edited cells. PRECISE-edited neutrophils retained normal function, including neutrophil extracellular trap formation, oxidative burst, and phagocytosis. Genome integrity analysis showed no genomic alterations or chromosomal aberrations, and only two off-target edits confined to non-coding intronic regions. In conclusion, PRECISE represents a translationally relevant base-editing strategy for ELANE-associated CN and CyN that addresses ELANE mutation heterogeneity.

Background Despite growing insights into the pathophysiology of non-union formation, failed fracture healing remains a major complication in trauma and orthopedic surgery. The transplantation of cancellous bone grafts represents the gold standard for the treatment of atrophic non-unions and large-scaled bone defects. Depending on the type of procedure and the available personnel, the bone grafts may be exposed to a significant period of intraoperative ischemia before the transplantation to the defect site. This ischemia may have detrimental effects on the quality and functionality of the grafts. Methods Therefore, we analyzed in this study the effects of different periods of ischemia (0, 30, 60 and 90 minutes) on oxidative stress, gene expression and viability of autologous bone grafts, to determine a critical ischemia time window for cancellous bone graft transplantation. Graft samples were harvested from 24 patients undergoing revision surgery due to bone healing failure. The samples were analyzed by mRNA profiler arrays, reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. Results An ischemia of 60 minutes or longer induced the expression of pro-inflammatory and stress-induced genes, such as CXCL8 , JUN and DUSP1 . This was associated with early cell stress within the grafts, as indicated by the presence of hypoxia-inducible factor (HIF)-1α-positive cells and an increased number of senescent p16-positive cells. Additional immunohistochemical analyses revealed a significantly higher number of apoptotic cleaved caspase-3-positive cells at 60 and 90 minutes of ischemia, demonstrating a compromised viability of the grafts. RT-PCR analyses revealed a shift from a pro-osteogenic towards a pro-chondrogenic extracellular matrix (ECM) gene expression profile, along with evidence for potentially compromised angiogenesis and hematoma formation at the later transplantation site. Conclusion Taken together, these findings indicate that periods of ischemia of 60 minutes or longer should be avoided during cancellous bone graft transplantation to preserve graft function and regenerative capacity.

Signaling lipids are key players in cellular processes. Despite their importance, no method currently allows their comprehensive monitoring in one analytical run. Challenges include a wide dynamic range, isomeric and isobaric species, and unwanted interaction along the separation path. Herein, we present a sensitive and robust targeted liquid chromatography-mass spectrometry (LC-MS/MS) approach to overcome these challenges, covering a broad panel of 17 different signaling lipid classes. It involves a simple one-phase sample extraction and lipid analysis using bioinert reversed-phase liquid chromatography coupled to targeted mass spectrometry. The workflow shows excellent sensitivity and repeatability in different biological matrices, enabling the sensitive and robust monitoring of 388 lipids in a single run of only 20 min. To benchmark our workflow, we characterized the human plasma signaling lipidome, quantifying 307 endogenous molecular lipid species. Furthermore, we investigated the signaling lipidome during platelet activation, identifying numerous regulations along important lipid signaling pathways. This highlights the potential of the presented method to investigate signaling lipids in complex biological systems, enabling unprecedentedly comprehensive analysis and direct insight into signaling pathways.